This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A major goal of the Klemke laboratory is to understand the molecular signaling mechanisms that control growth cone and neurite formation. The ability of neuronal cells to extend and retract neurites is important for proper brain development and is important for spinal cord regeneration after injury. However, it has been difficult to study this process because it has not been possible to biochemically isolate the neurite for protein analysis. Recently, we developed a new biochemical method using microporous technology to purify the neurite in large scale from neuronal cells (BioTechniques. 2003;35:254-256). This novel system will allow us to perform large-scale proteomics to identify the key regulatory proteins that facilitate growth cone formation and neurite extension and retraction in response to chemoattractants or chemorepulsion agents, respectively. We will use mouse and human neuroblastoma cells for these studies as they readily extend/retract neurites. Initial analysis using these cells revealed that phosphotyrosine (PY) proteins are highly activated and enriched in the neurite fraction compared to the soma. Pharmacological inhibition of tyrosine phosphorylation inhibits growth cone formation and neurite extension indicating that complex signaling cascades control this process through modulation of PY networks. Therefore, our objective is to characterize the PY proteins (neurite phosphoproteome) responsible for growth cone formation and neurite extension/retraction. PY proteins from isolated somas and extending or retracting neurites will be immunoaffinity purified with anti-PY antibodies and/or enriched for phosphopeptides using an IMAC column and then analyzed using the Center's high sensitivity, high resolution LC-MS/MS capabilities to identify key proteins and determine the specific locations of the phosphorylated residues. Functional testing will then be performed using siRNA protein knockdown and site directed mutagenesis followed by cell-based assays and animal models of neurite formation established in our laboratory. Information gained from these experiments will be analyzed using bioinformatics and computer modeling to reveal potential phosphotyrosine networks that contribute to neurite formation during spinal cord injury and neuronal degeneration. Results from our study will provide valuable information on the phosphosignals that control neurite formation and provide targets for therapeutic treatment of neurodegenerative diseases as well as spinal cord regeneration. Specific Aims: 1. To identify PY proteins and their specific sites of tyrosine phosphorylation in extending or retracting neurites. 2. To functionally test identified PY proteins using siRNA protein knockdown and site directed mutagenesis of key phosphotyrosine sites (as identified by MS) followed by cell-based assays and animal models of neurite formation established in our laboratory. 3. To map the putative signaling cascades and develop functional relationships among the PY proteins using bioinformatics and computer modeling systems. Methods for Specific Aim 1: An initial "bottom-up" analysis of immunopurified neurite PY proteins will be performed using PNNL's high sensitivity and high resolution LC-MS/MS. Immunopurified PY proteins will be subjected to tryptic digestion, peptide purification, and an off-line strong cation exchange separation coupled to LC-MS/MS analysis for the identification of PY proteins. To identify the specific tyrosine phosphorylation sites on the phosphoproteins, a portion of the tryptic digest will be passed through an IMAC column to enrich for phosphopeptides present in the digest following methyl etherification of the peptides. This phosphopeptide enriched sample will then be analyzed by LC-MS/MS to determine the identities of the phosphopeptides and the specific locations of the phosphorylated residues which can then be targeted for achieving specific aim #2: functional testing of key PY proteins using siRNA-directed knockdown approaches and/or site directed mutagenesis strategies. Information gained from aims 1 and 2 will then be analyzed using bioinformatics and computer modeling to reveal potential phosphotyrosine networks that contribute to neurite formation, which is important for proper brain development and spinal cord regeneration after injury.